Leuconostoc holzapfelii sp. nov., isolated from Ethiopian coffee fermentation and assessment of sequence analysis of housekeeping genes for delineation of Leuconostoc species

Abstract

A Gram-positive, ovoid lactic acid bacterium, strain LMG 23990 T

Molecular identification and typing of lactic acid bacteria associated with the production of two artisanal raw milk cheeses

“…Subsets of isolates recovered on a given medium from a given sample type (milk, starter, curd or cheese at different ripening times) collected during a given sampling campaign were included for separate dendrogram construction. In each of these dendrograms, (GTG) 5 -PCR clusters were delineated based on visual comparison of band patterns and a minimum Pearson similarity coefficient of 90.0%. For each (GTG) 5 -PCR cluster, one or two representative isolates were selected.…”

“…In each of these dendrograms, (GTG) 5 -PCR clusters were delineated based on visual comparison of band patterns and a minimum Pearson similarity coefficient of 90.0%. For each (GTG) 5 -PCR cluster, one or two representative isolates were selected. This way, the initial set of 734 isolates was reduced to a collection of 298 genotypically unique isolates.…”

“…This way, the initial set of 734 isolates was reduced to a collection of 298 genotypically unique isolates. This selection was subsequently subjected to DNA extraction using an adapted protocol of Pitcher et al [11,16] and reanalyzed using (GTG) 5 -PCR. The new (GTG) 5 -PCR profiles were then compared with an inhouse BioNumerics reference database for identification.…”

“…16S rRNA gene sequencing is one of the most commonly used techniques for identification and classification of LAB species, but fails to discriminate between phylogenetically closely related LAB species or between subspecies [6]. A number of recent studies indicate that alternative gene targets for sequencing such as the RNA polymerase B subunit gene, rpoB, and the phenylalanyltRNA synthase gene, pheS, display a higher taxonomic resolution than the 16S rRNA gene within several LAB genera [5,14,18]. For differentiation at strain level, molecular fingerprinting techniques such as repetitive DNA element PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) analysis [19] have been well validated for LAB.…”

“…In the present study, rep-PCR using the (GTG) 5 -primer and AFLP were used as molecular fingerprinting tools to study the genotypic diversity and persistence of LAB strains throughout successive productions and ripening of two Flemish artisanal raw milk cheeses.…”